Motor Proteins, Cytoskeleton, and Cell Motility
Key words: Molecular motors, filopodia, cytoskeleton, cell motility, myosin-X
Molecular basis of cell movement
The cytoskeleton and cell movement underlie a host of processes important for human health and disease, including angiogenesis, the development of the nervous system, and the metastasis of cancer cells. Our goal is to elucidate the molecular basis of cell movement by investigating the properties and cell biological functions of motor proteins. We are particularly interested in the actin cytoskeleton and the actin-based motors known as unconventional myosins. Humans express genes for 24 different unconventional myosins, and mutations in these proteins lead to a variety of diseases, including several forms of deafness. Much of our current research focuses on myosin-X (Myo10), an unconventional myosin that is broadly expressed in vertebrate tissues. We have shown that Myo10 has central roles in the formation and function of filopodia, the finger-like, actin-based cellular extensions thought to act as sensors that guide the growth of nerves and blood vessels. In addition to acting as a molecular motor that binds to and moves along actin filaments, Myo10 also binds to the guidance receptor Deleted in Colorectal Cancer (DCC) and to integrins, a key class of cell adhesion receptors. Myo10 also binds to PIP3 and is likely to function as a filopodial effector for the important signaling molecule PI3-kinase. Myo10 is also a key component of invadopodia, which are actin-based cell extensions that facilitate the spread of cancer cells. In addition to these roles in filopodia and related actin-based structures, Myo10 can also bind to microtubules and is required for proper positioning of mitotic and meiotic spindles.
Our research with myosin-X and filopodia utilizes a variety of techniques including cell culture, live cell imaging, TIRF, molecular biology, and biochemistry. In addition to using simple cellular models such as HeLa and COS-7 cells, we are also investigating the functions of Myo10 in more specialized cell types. These include epithelial cells, where we have found that Myo10 localizes to basolateral filopodia, endothelial cells, where Myo10 is likely to function in the endothelial tip cells that guide growing blood vessels, and neurons, where we recently found that a "headless" form of Myo10 is expressed in neuronal stem cells. We also investigate a variety of other unconventional myosins, including brain myosin-V (Myo5a), where we did much of the initial characterization its fundamental properties such as step size and processivity. Our discovery of myosin-Vc (Myo5c) revealed that it functions as a class V myosin of exocrine secretory granules in secretory epithelia such as breast, prostate, and lacrimal gland. Our discovery and initial characterization of human myosin-XIX (Myo19) revealed that this novel class of myosin localizes to mitochondria and provides a potential molecular mechanism for actin-dependent mitochondrial movement and localization in animal cells.
|Scanning EM showing that overexpression of GFP-Myo10 leads to a massive increase in dorsal filopodia in COS-7 cells.|
What are the components of the filopodial tip complex and what does it do?
The striking localization of Myo10 to the tips of filopodia, as well as the localization of other myosins to the tips of related actin-based structures such as intestinal microvilli and inner ear stereocilia (the sensors for human hearing and balance), raises basic questions about the composition and functions of the material at the tips of these structures. We hypothesize that Myo10 is a central component of a filopodial tip complex that functions in actin polymerization, cell adhesion, and cell signaling, and one of our current goals is to identify the other components of this tip complex. Since the filopodial tip also appears to represent a specialized site of cell adhesion and interaction with other cells, we are also investigating how the composition of the filopodial tip changes as filopodia extend, retract, and interact with their surroundings.
How does Myo10 induce filopodia?
Although we have shown that overexpressing Myo10 massively increases filopodia and that knocking down Myo10 decreases filopodia, the mechanisms by which Myo10 induces filopodia remain unclear. The fact that mutations in distantly related MyTH-FERM myosins such as Myo7a and Myo15a lead to defects in stereocilia and human diseases such as Usher Syndrome also indicates that the MyTH-FERM myosins play key roles in the formation and function of cellular protrusions, but exactly how this phylogenetically ancient family of motor proteins acts is unclear.
One of the most fascinating features of Myo10 is our discovery that that it exhibits novel forms of motility within filopodia. In addition to maintaining itself at the tips of filopodia as they extend and retract, puncta of GFP-Myo10 can also undergo forward and rearward movements within filopodia. Using a sensitive TIRF (total internal reflection fluorescence) microscope, we have also been able to observe extremely faint particles of GFP-Myo10 moving very rapidly (500-1000 nm/s) forward in filopodia, leading us to hypothesize that these movements of Myo10 molecules represent a previously unrecognized system of intracellular transport that occurs at the single molecule level. This discovery of the intrafilopodial motility of Myo10 raises a host of questions, including how this transport is regulated, what its cargos are, and whether other myosins power analogous forms of transport in microvilli and stereocilia.